Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma
Abbatiello, Susan E., Schilling, Birgit, Mani, D.R., Zimmerman, Lisa J., Hall, Steven C., Maclean, Brendan, Albertolle, Matthew, Allen, Simon, Burgess, Michael, Cusack, Michael P., Ghosh, Mousumi, Hedrick, Victoria, Held, Jason M., Inerowicz, H. Dorota, Jackson, Angela, Keshishian, Hasmik, Kinsinger, Christopher R., Lyssand, John, Makowski, Lee, Mesri, Mehdi, Rodriguez, Henry, Rudnick, Paul, Sadowski, Pawel, Sedransk, Nell, Shaddox, Kent, Skates, Stephen J., Kuhn, Eric, Smith, Derek, Whiteaker, Jeffery R., Whitwell, Corbin, Zhang, Shucha, Borchers, Christoph H., Fisher, Susan J., Gibson, Bradford W., Liebler, Daniel C., MacCoss, Michael J., Neubert, Thomas A., Paulovich, Amanda G., Regnier, Fred E., Tempst, Paul, & Carr, Steven A. (2015) Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma. Molecular and Cellular Proteomics, 14(9), pp. 2357-2374.
There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.
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|Item Type:||Journal Article|
|Keywords:||Absolute quantification, Assay development, Multiple reaction monitoring, Quantification, Stable isotope dilution, Targeted mass spectrometry, abundant protein depletion, analytical validation, labeled protein internal standards, multiplexed|
|Divisions:||Current > Schools > School of Earth, Environmental & Biological Sciences
Current > Institutes > Institute for Future Environments
Current > QUT Faculties and Divisions > Science & Engineering Faculty
|Copyright Owner:||Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.|
|Copyright Statement:||This research was originally published in Molecular and Cellular Proteomics. Abbatiello, Susan E. et al. Large-scale inter-laboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma. Molecular and Cellular Proteomics. 2015. Vol:pp-pp. © the American Society for Biochemistry and Molecular Biology|
|Deposited On:||06 Apr 2015 23:13|
|Last Modified:||17 May 2016 02:40|
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